This invention relates to a mutant loxP site and applications thereof. More particularly, the present invention relates to the mutant loxP site, in which a specific recombination between the mutant loxP site and a wild-type loxP site can not occur, but a specific recombination between mutant loxP sites can occur in each other, and gene replacement using the said mutant loxP site.
It is not so easy to integrate any gene into specific sites of animal virus or chromosome of animal cells of the higher eucaryotes or to delete specific gene therefrom. A conventional method for gene integration into the specific site of chromosome of animal cells is, for example, that cells are transformed with plasmid DNA, to which DNA having the same site with the site of chromosome to be intended to integrate is ligated with both sites of the objective gene, to obtain the cells, to which the objective gene is integrated by homologous recombination. Frequency of homologous recombination is, however, extremely low. To that end, the objective gene and drug resistant gene should be simultaneously integrated and selected by drug. Consequently, several months have to be required to obtain the objective cells. Further, although preparation of recombinant animal virus, to which the objective gene is integrated, is slightly easier than the previously described case of chromosome of cells, however even in case that, for example, the recombinant adneovirus is constructed, various treatments including homologous recombination by using plasmids, to which objective gene is integrated, as well as cloning, selection and growth of the recombinant virus are required (Bett et al., Proc. Natl. Acad. Sci., 91: 8802-8806, 1999 and Miyake et al., ibid. 93: 1320-1324, 1996).
One of reasons why the gene manipulation of specific sites in chromosome of animal cells and the construction of recombinant virus are difficult is using homologous recombination with low frequency. Contrary to that, if it can be used enzymes, which can specifically recognize DNA Sequence, as like restriction enzymes used for construction of plasmid or bacteriophage, it is expected to improve the efficiency of the gene manipulation on cell chromosome. Example of such the enzyme is recombinase Cre derived from bacteriophage P1 of E. coli. 
Cre is a specific DNA recombinase, which recognizes specific nucleotide sequence (loxP site) and conducts total processes including DNA strand cleavage, strand exchange and ligation of each DNA strand within this site (Sternberg et al., J. Mol. Biol., 150: 467-468, 1981; Abremski et al., J. Biol. Chem., 259: 1509-1514, 1984; and Hoess et al., Proc. Natl. Acad. Sci., 81: 1026-1029, 1984). In case that two loxP sites of the same direction exist within the same DNA molecule, DNA sequence between them is excised to form circular molecule (DNA excision reaction), or on the contrary in case that two loxP sites exist in the different DNA molecules, and the one is a circular DNA, the circular DNA is inserted into the other DNA molecule through loxP site (insertion reaction). Although Cre and loxP site were found in bacteriophage, the specific DNA recombination reaction is known to function not only in the procaryotes but also in the eucaryotes including animal cells and in the animal viruses. Examples of excision reactions are cultured animal cells (Sauer et al., Nucleic Acids Res., 17: 147-161, 1989 and Kanegae et al., Gene, 181: 207-212, 1996), animal viruses (Sauer et al., Proc. Natl. Acad. Sci., 85: 5166-5170, 1988; Anton et al., J. Virol., 69: 4600-4606, 1995; and Kanegae et al., Nucleic Acids Res., 23: 3816-3821, 1995), and transgenic mice (Lakso et al., Proc. Natl. Acad. Sci., 89: 6232-6236, 1992; Orban et al., ibid., 89: 6861-6865, 1992; Gu et al., Cell, 73: 1155-1164, 1993 and Gu et al., Science, 265: 103-106, 1994).
In addition, if the insertion reaction is applied, any gene can be inserted into the chromosome of animal cells or viral genome, in which loxP site exists previously, but the frequency of insertion is extremely low (Fukushige et al., Proc. Natl. Acad. Sci., 89: 7905-79029, 1992 and Sauer et al., Proc. Natl. Acad. Sci., 84: 9108-9112, 1987), consequently it is not practicable. Because, the insertion and excision are irreversible reactions, consequently if two loxP sites are existed in the identical DNA molecule as a result of insertion reaction, the excision reaction immediately occurs, moreover a reaction equilibrium lies overwhelmingly so far to the excision reaction.
In order to increase frequency of the insertion reaction, trials on using loxP site (mutant type), which is different from the original nucleotide sequence of loxP site (wild-type), were performed. The loxP site consists of DNA sequence of 34 bp (SEQ ID NO: 1), as shown below in both the 5xe2x80x2 to 3xe2x80x2 and the 3xe2x80x2 to 5xe2x80x2 direction. Among them, 8 bp sequence between two 13 bp inverted repeats is designated as spacer region, and recombination of DNA strand is known to be carried out within the spacer region (Hoess et al., J. Mol. Biol., 181: 351-362, 1985).
It was shown that the specific DNA recombination reaction between loxP site (mutant loxP site), in which a base at position 7 in the spacer region is substituted from G (guanine) to A (adenine), and wild-type loxP sequence can not occur, but the specific DNA recombination reaction between two mutant loxP sites can occur (Hoess et al., Nucleic Acids Res., 14: 2287-2300, 1986).
Trials that a gene located between mutant loxP site and wild-type loxP site in the DNA molecule is inserted between mutant loxP site and wild-type loxP site in the other DNA molecule or replaced by the other gene between them, are carried out. Examples of these trials are replacement of a gene on the plasmid vector by a gene on the bacteriophage gene (Waterhouse et al., Nucleic Acids Res., 21: 2265-2266, 1993), insertion of a gene on the phagemid vector to the plasmid vector (Tsurushita et al., Gene, 172: 59-63, 1996) and replacement of a gene on the plasmid vector by a gene on the chromosome of animal cells (Bethke et al., Nucleic Acids Res., 25: 2828-2834, 1997).
These trials were, however, performed by using only one mutant loxP site, i.e. the loxP site in which a base at position 7 of the spacer region was substituted from G (guanine) to A (adenine) (mutant loxP site), and the fact that whether it is preferable or not, is unknown, because the recombination reaction between the mutant loxP site in the said sequence and the wild-type loxP site does not occur. Further, in the above all three trials, the experimental systems, in which the drug resistant gene itself or the drug resistant gene together with objective gene is inserted and as a result, the recombinants having DNA molecules accompanied with the objective recombination can only acquire drug resistance and amplyfy, are used. Consequently, even if efficiency of the actual gene insertion (gene replacement) is low due to the recombiantion between the loxP site with incomplete mutation and the wild-type loxP site, such the experimental result is biased by the selection with drug resistance, and the apparent reaction efficiency may possibly be expressed too high.
Actually, as a result of direct and quantitative measurement found by us, in the mutant loxP site with substitution from G to A at position 7, a recombination reaction between it and the wild-type loxP sequence occurs with frequency of approximately 5%, which shows incomplete mutation of the loxP site.
As explained hereinabove, in the prior art, a technique for performing gene replacement in the chromosome in animal cells using mutant loxP site and wilt-type loxP site has tried, but its efficiency was not sufficient.
An object of the present invention is to provide a mutant loxP site wherein, in the presence of recombinase Cre, recombination with a wild-type loxP site can not occur, and recombination between two mutant loxP sites having the identical sequence can occur at the almost same efficiency of the recombination between two wild-type loxP sites. Further object of the present invention is to provide a method for gene integration or gene replacement with high efficiency in higher eukaryote including animal cells by the combination of the wild-type loxP site and the mutant loxP site, or the combination of the mutant loxP sites having different sequences in each other. More further object of the present invention is to provide application of such the methods for gene transfer to animal and plant cells, construction of recombinant viruses, gene manipulation in the animal and plant bodies, and the like.
We have studied a mechanism of recombinase Cre-dependent recombination between two loxP sites and identified nucleotide sequence of loxP site essential for the reaction, as a result of preparing mutant loxP sites with possible single base substitution for all of 8 bases in the spacer region of loxP site and studying the reactivity by means of very sensitive assay method. Accordingly, we have identified, on the basis of these findings, the mutant loxP site, in which a recombination between two mutant loxP sites having the identical sequence can occur with nearly equal efficiency of the recombination between two wild-type loxP sites, and a recombination between the mutant loxP site and the wild-type loxP site or between the mutant loxP sites having different sequence can not occur. Further we have succeeded, as a result of combining these loxP sites, to integrate a gene with extremely high efficiency into chromosome of animal cells. The present invention has been completed based on these findings and as a result of further progress of studies.
Accordingly, the gist of the present invention is as shown in the following (1)-(2 1);
(1) A mutant loxP site having following properties:
(a) a nucleotide sequence wherein, in a wild-type loxP site of the following formula (SEQ ID NO: 1) derived from E. coli P1 phage, at least one of the bases consisting of second (T), third (G), fourth (T) and fifth (A) bases, and at least one of the bases consisting of sixth (T) and seventh (G) bases within the 8 bases in the central part of the sequence (spacer region) are substituted by different bases, and regions except for the spacer
Region are optionally substituted by any base;
(b) a specific recombination between said mutant loxP site and the wild-type loxP site can not occur even in the presence of recombinase Cre; and
(c) a specific recombination between the mutant loxP sites having identical nucleotide sequences can occur in the presence of recombinase Cre.
(2) A mutant loxP site having following properties:
(a) a nucleotide sequence wherein, in a wild-type loxP site of the following formula (SEQ ID NO: 1) derived from E. coli P1 phage, a base selected from the group consisting of second (T), third (G) and fourth (T) bases is substituted by a different base, and regions except for the spacer region are optionally substituted by any base;
(b) a specific recombination between the said mutant loxP site and the wild-type loxP site can not occur even in the presence of recombinase Cre; and
(c) a specific recombination between the mutant loxP site having identical nucleotide sequences can occur in the presence of recombinase Cre.
(3) The mutant loxP site according to (1) or (2) above, wherein the specific DNA recombination between the mutant loxP site and another mutant loxP site having different nucleotide sequence can not occur in the presence of recombinase Cre.
(4) The mutant loxP site according to (1) above, wherein the nucleotide sequence is expressed by SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 42, or SEQ ID NO: 49.
(5) A DNA comprising the mutant loxP site according to any one of (1) to (4) above.
(6) A DNA comprising at least one wild-type loxP site and at least one mutant loxP site according to (1) or (2) above.
(7) The DNA according to (6) above wherein a desired gene is inserted between the wild-type loxP site and the mutant loxP site.
(8) A DNA comprising at least two mutant loxP sites having different nucleotide sequence in each other according to (3) above.
(9) The DNA according to (8) above wherein a desired gene is inserted between two mutant loxP sites having different nucleotide sequence in each other.
(10) A cell which is transformed by DNA according to any one of (6) to (9) above.
(11) A method for replacing gene comprising reacting DNA (a) and DNA (b) hereinbelow in the presence of recombinase Cre and obtaining DNA (c) hereinbelow:
(a) a DNA comprising of a wild-type loxP site, a gene A and a mutant loxP site according to (1) or (2) above, in this order;
(b) a circular DNA comprising of a wild-type loxP site, a gene B and the same mutant loxP site as DNA (a) in this order; and
(c) DNA in which a gene A is replaced by a gene B in DNA (a) wherein each of gene A and gene B is any gene selected from the genes different in each other.
(12) A method for replacing gene comprising reacting DNA (a) and DNA (b) hereinbelow in the presence of recombinase Cre and obtaining DNA (c) hereinbelow:
(a) a DNA comprising of two mutant loxP sites having different nucleotide sequences in each other according to (3) above (mutant loxP site 1 and mutant loxP site 2) and gene A, arranged in the order of mutant loxP site 1/gene A/mutant loxP site 2;
(b) a circular DNA comprising of the mutant loxP site 1, the gene B and the mutant loxP site 2, in this order; and
(c) a DNA in which gene A is replaced by gene B in DNA (a) wherein each of gene A and gene B is any gene selected from the genes different in each other.
(13) The method according to (11) or (12) above wherein the gene B is not a functional gene.
(14) The method according to (11) or (12) above wherein the gene A is not a functional gene.
(15) The method according to any one of (11) to (14) above wherein DNA (a) is chromosomal DNA of cells and DNA (b) is plasmid DNA or DNA of DNA of double stranded circular DNA virus.
(16) The method according to any one of (11) to (14) above wherein DNA (a) is chromosomal DNA of cells and DNA (b) has properties to be converted intracellularly to double-stranded circular DNA.
(17) The method according to any one of (11) to (14) above wherein DNA (a) is chromosomal DNA of double-stranded DNA viruses and DNA (b) is plasmid DNA or DNA of DNA of double-stranded circular DNA virus.
(18) The method according to any one of (11) to (14) above wherein DNA (a) is chromosomal DNA of double-stranded DNA viruses and DNA (b) has properties to be converted intracellularly to double-stranded circular DNA.
(19) The method according to (17) or (18) above wherein double-stranded DNA virus of DNA (a) is adenovirus.
(20) A transgenic animal having DNA according to any one of (6) to (9) above on the chromosome.
(21) A pharmaceutical product comprising DNA according to any one of (6) to (9) above.